The process of X-inactivation in mammals occurs by the formation of facultative heterochromatin, a phenomenon central to normal development and abrogated in some cancers. In this process, an accumulation of stable XIST RNA structurally associates with one X chromosome in females and initiates a cascade of chromosome remodeling that silences the inactive X (Xi), forming a heterochromatic Barr Body. We now need to know how XIST RNA localizes to and "paints" its parent chromosome, and how this leads to the structural transformation and condensation of a whole chromosome. Our approach to these questions utilizes molecular, biochemical and structural analyses, coupled with bioinformatics of genomic sequence organization. Our aims deal with distinct but inter-related aspects of the functional and structural transformation of the chromosome, focusing on the interaction of XIST RNA with the chromosome and the potential role genomic repeat sequences. Aim 1 builds on our recent success in manipulating XIST RNA localization, to better understand the regulation of XIST binding, specific factors involved, and extend strong preliminary results on specific histone modifications, as well as heterochromatin factors and scaffold attachment factors implicated in Xi. The factors involve may provide insight into broad heterochromatic instability in cancer, of which Xi/XIST defects may be one hallmark. Aim 2 investigates a novel model for silencing of an entire chromosome, where XIST RNA is not acting at a local or individual gene level, but has a more architectural relationship with the whole interphase chromosome territory, particularly with an inner core enriched in repeat elements. In this model, XIST first interacts with the repeat-rich regions of the X chromosome, nucleating a heterochromatic core that then propagates to the more peripheral protein coding genes. In Aim 3, the relationship of sequence context to escape from silencing is examined in a region that consistently escapes silencing, using a systematic transgene approach, combined with bioinformatics and molecular cytological analyses. Our studies will focus largely on human X inactivation, which has been less well studied, using somatic cell, transgene and human ES cell models. Understanding what establishes and maintains human Xi heterochromatin has relevance to heterochromatic instability in cancer as well as formation of facultative heterochromatin in embryonic cells, and thus our studies have significant clinical implications.